The present invention relates to a process for detection of the presence of biological matters of bovine origin in a sample of organic matter.
It also relates to oligonucleotides for carrying out this process.
In 1986, bovine spongiform encephalopathy (BSE), or xe2x80x9cmad cowxe2x80x9d disease, showed up for the first time in British bovine livestock. Since then, more than 100,000 clinical cases have been identified in cattle. This disease, which is present in Great Britain, has as endemic evolution. It has also been observed in various European countries: Ireland, Switzerland, France, Denmark, Germanyxe2x80x94where it evolves sporadically.
The clinical picture of the disease is known. The infectious agent responsible, called xe2x80x9cprionxe2x80x9d is characterized by, among other properties, an extreme resistance to conventional decontaminating agents, such as heat, radiation or detergents. Recent studies furthermore have demonstrated the very high resistance of the infectious agent under xe2x80x9cnaturalxe2x80x9d conditions, and in particular its persistence in pastures. The infectiousness can therefore persist in the ground for at least 3 years.
One of the modes of transmission of the infectious agent is ingestion of contaminated foods, and this transmission can furthermore be from one animal species to another.
Analysis of the epidemiological data has enabled the origin of the English epidemic to be discovered, this being due to the infectious agent contaminating the meat meals and bone meals (MBM) used to manufacture food additives intended for feeding dairy cows. These meals are the by-products of quartering shops arising from treatment of carcasses and waste originating from abattoirs.
The structures of xe2x80x9cprionxe2x80x9d is not known to this day, and there is no test for detecting it.
It therefore important to determine whether organic matter contains biological matters of bovine origin and is capable of containing xe2x80x9cprionsxe2x80x9d.
In the agricultural food field, the characterization of animal species initially used biochemical techniques for analysis of proteins (BARA et al. 1992, Trends in Food Science and Technology, 3, 69-72; SOTELO et al. 1993, Trends in Food Science and Technology, 4, 395-401; Hernandez et al., Food and Agricultural Immunology, 6, 95-104). However, these methods are not very specific (electrophoresis), or are incompatible with denaturation of the samples to be analysed (immunoanalysis). They are now progressively being replaced by the techniques of DNA analysis, this molecule being less sensitive than proteins to denaturing physico-chemical conditions.
Identification of the main animal species of interest was first carried out by the technique of hybridization of nucleic probes (BUNTJER et al. 1995, Zeitschrift fuer Lebensmittel Untersuchung und Forschung 201 (6): 577-582; MEYER et al., 1994, Fleischwirtschaft 74 (11) 1237-1238; TSUMURA et al. 1992, Journal of Japanese Society of Food Science and Technology 39 (1) 60-63; EBBEHOJ and THOMSEN, 1991, Meat Science 30 (4): 359-366; BAUER et al., 1987, Archiv fuer Lebensmittelhygiene 38 (6): 172-174; EBBEHOJ and THOMSEN, 1991, Meat Science 30 (3): 221-234).
This technology, which is tricky to use, has now been superseded by the PCR (polymerase chain reaction) method, which has been used to characterize biological matters originating from various animal species.
The only methods for amplification by PCR described in beef (Bos taurus) use amplification of the mitochondrial DNA (mtDNA) region which codes for a cytochrome by means of PCR primers which recognize sequences conserved in species of vertebrates, and then their characterization by means of restriction enzymes (RFLP) or by sequencing (MEYER et al. 1995, Journal of AOACxe2x80x94International 78 (6): 1542-1551; CHIKUNI et al. 1994, Animal Science and Technology, 65 (6): 571-579; GUGLICH at al. 1994, J. Forensic. Sci. 39 (2): 353-61).
The organization and complete sequence of bovine mitochondrial DNA (mtDNA) are known (ANDERSON et al. 1982, J. Mol. Biol. 156 (4): 683-717). On the basis of these data, several works have been dedicated to the study of the genetic variability of the mitochondrial genome of domestic Bovidae by analysis of the restriction polymorphism (CHEN et al. 1995, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 111 (4): 643-649; KIKKAWA et al. 1995, Biochem. Genet. 33; (1-2): 51-60; BRADLEY et al. 1994, Anim. Genet (4): 265-271; AMANO et al. 1994, Anim. Genet. 25 (1): 29-36; SUZUKI et al. 1993, Anim. Genet 24 (5): 339-343: Lan et al. 1993, I. Chuan. Hsueh Pao 20 (5): 419-425; BHAT et al. 1990, Biochem. Genet 28 (7-8): 311-318; LOFTUS et al., 1994, Anim. Genet. 25:265-271) or by sequencing (LOFTUS et al. 1994, Proc. Natl. Sci. USA 91 (7): 2757-2761; RON et al. 1993, Anim. Genet. 24 (3): 183-186; BRADLEY et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5131-5135; BAILEY et al., 1996, Proc. R Soc. Lond. B, 263:1467-1473).
Partial sequencing of the control region of bovine mtDNA has furthermore been carried out for various European, African and Indian bovine breeds (LOFTUS et al., 1994 Proc. Natl. Sci. USA 91 (7): 2757-2761; BRADLEY et al., 1996, Proc. Natl. Acad. Sci. USA 93: 5131-5135; BAILEY et al., 1996, Proc. R. Soc. Lond. B, 263:1467-1473).
It thus emerges from studying the prior art that works on analyses of the DNA of certain species and bovine breeds have already been carried out. Nevertheless, none of the documents of the prior art describes a specific and sensitive method for amplification of bovine DNA which enables traces of biological matters of bovine origin to be identified and can be used for all bovine breeds and in organic matter having widely varying compositions.
In fact, the known techniques for identification of bovine DNA (for example analysis of genomes by RFLP or PCR-RFLP carried out on portions of sequences which vary little) have disadvantages. By these methods, which are not very specific, it is often difficult to analyse mixtures of DNA originating from different species because of the large number of bands and the difficulties in interpretation associated with this characteristic.
The low sensitivity of some of these techniques does not enable the presence of organic matter of bovine origin in widely diverse organic substrates to be demonstrated in a reliable manner. These methods cannot be used if the DNA present in the sample is degraded in the form of small fragments having a size of less than about 500 base pairs.
The problem of identification of organic matter originating from any of the bovine breed is particularly crucial, since bovine spongiform encephalitis is not limited to European bovine breeds, but extends to African and Indian breeds (Bos taurus and Bos indicus).
The Applicant has thus concerned himself with solving these problems.
It has been found that the presence of biological matter of bovine origin could be detected in a specific and simple manner and with a high sensitivity, whatever the bovine breed (Bos taurus and Bos indicus), in samples of organic matter by amplifying a defined sequence of the bovine genome in a specific manner.
In its most general form, the present invention thus relates to a process for obtaining a fragment of bovine DNA which has a defined size and sequence and is specific to bovines, and in particular the species Bos taurus and Bos indicus, from a sample of organic matter, and a process by which a defined sequence of the bovine genome present in bovine genomes but absent from the genomes of other animal species is amplified by a polymerase chain reaction.
The present invention also relates to a process for detection and identification of the presence of biological matter of bovine origin in a sample of organic matter, characterized in that the presence of DNA of bovine origin is determined in the said organic matter by amplification of a specific DNA sequence of the bovine genome.
Organic matter is understood as meaning any solid or liquid matter which is assumed to have at least partly a biological origin.
The DNA sequence is advantageously of mitochondrial origin. The choice of a mitochondrial sequence is particularly advantageous, since in an animal cell there are about 100 to 1,000 copies of mitochondrial DNA for one copy of nuclear DNA. In the event of degradation of the DNA, the probability of detecting the mitochondrial DNA is thus much higher than the probability of detecting the nuclear DNA. In addition, the mitochondrial DNA is more resistant to degradation than the nuclear DNA. The mitochondrial DNA can therefore be detected more reliably in organic matter in which the DNA is subjected to various physical factors (heat, pressure . . . ), chemical factors (hydrolysis, oxidation . . . ) or biochemical factors which tend to degrade it.
This characteristic proves to be particularly important if the intention is to detect the presence of biological matter of bovine origin in organic matter which has been subjected to several transformations, for example in cosmetics, in agricultural foods, such as the meals used for feeding cattle, composts, manures and dungs etc. . . .
The invention is also advantageous for detection of the presence of biological matters of bovine origin in the following organic substrates: raw, smoked or cooked meats, pellets, blood and products based on blood, milk and products based on milk, bone and products based on bone, hides, skins, ivories, furs, horns and products based on horn, guano, faeces, semi-liquid manures, liquid manures, gelatine and products based on gelatine, and cosmetic and agricultural food products.
The invention relates to specific mitochondrial DNA fragments of the bovine genome having sizes ranging from about 500 base pairs to about 100 base pairs, in particular about 152 to 480 base pairs, and having a sequence identical to the extent of at least 80%, and preferably to the extent of at least 90%, to homologous regions of the sequence of the control region of the mitochondrial DNA determined by ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717 and in particular ranging from position 15,824 to position 171, these positions being determined according to the complete mitochondrial DNA sequence of beef, which comprises 16,338 nucleotides, determined by ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717.
Preferably, the DNA amplification is carried out by the polymerase chain amplification method (PCR), comprising repetition of a cycle made up of the following stages:
Heating of the DNA extracted from the sample of organic matter such that the DNA is separated into two monocatenated stands.
Hybridization of oligonucleotide primers with the monocatenated DNA strands at an appropriate temperature and
Elongation, of the oligonucleotide primers by a polymerase at an appropriate temperature.
Particularly preferably, one of the primers is an oligonucleotide having a sequence identical to the extent of at least 80%, preferably to the extent of at least 90%, and advantageously to the extent of at least 95%, to an oligonucleotide made up of a sequence of about 15 to 25 nucleotides, in particular about 20 to 25 nucleotides, contained in the following sequence SEQ ID No. 1 (positions 136 to 178 according to ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717):
TAATGTCCATGCTTATCATTATGCTGGTGCTCAAGATGCAGTT
This first primer can be, in particular, an oligonucleotide or a mixture of oligonucleotides comprising the following sequence SEQ ID No. 2 (positions 152 to 166 according to ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717):
YTATCATTATGCTGG
in which Y is T or C.
It is preferably an oligonucleotide or a mixture of oligonucleotides having the following sequence SEQ ID No. 3 (positions 152 to 171 according to ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717):
CATGCYTATCATTATGCTGG PBR6)
in which Y is T or C.
The second primer is an oligonucleotide having a sequence identical to the extent of at least 80%, preferably to the extent of at least 90%, and advantageously to the extent of at least 95%, to an oligonucleotide made up of a sequence of about 15 to 25 nucleotides, in particular about 20 to 25 nucleotides, contained in the following sequence SEQ ID No. 4 (positions 16,015 to 16,060 according to ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717):
ATTATATGCCCCATGCATATAAGCAAGTACATGACCTCTATAGCAG
Such an oligonucleotide is preferably that comprising the following sequence SEQ ID No. 5 (positions 16,034 to 16,048 according to ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717):
TAAGCAAGTACATGA
or preferably that having the following sequence SEQ ID No. 6 (positions 16,029 to 16,048 according to ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717):
GCATATAAGCAAGTACATGA (PBF9)
Each of the oligonucleotides SEQ ID No. 1 to 3 is used as a pair with one or other of oligonucleotides SEQ ID No. 4 to 6. The most advantageous pair of primers is the pair SEQ ID No. 3 and SEQ ID No. 6.
According to a particularly advantageous embodiment of the present invention, at least part of the hybridization stages of the cycles which make up the amplification reaction is carried out at a temperature of about 50xc2x0 C. to 58xc2x0 C., in particular 50xc2x0 C. to 55xc2x0 C. In add the Applicant has found that a temperature of about 51xc2x0 C. was particularly suitable for obtaining a specific amplification.
Such an embodiment enables a high amplification specificity to be obtained.
The temperatures of the stages of separation of the strands and elongation are advantageously about 94xc2x0 C. and 72xc2x0 C. respectively.
The process described above is specific since it gives no amplification reaction which can be detected in the presence of DNA of other than bovine origin (Bos taurus and Bos indicus). The use of the primers SEQ ID No. 1 to SEQ ID No. 6 gives rise only to a DNA fragment of about 480 base pairs. The present invention also relates to this oligonucleotide fragment.
It advantageously has a sequence identical to the extent of at least 80%, preferably 90%, and advantageously 95%, to the following sequence SEQ ID No. 8 (positions 16,029 to 171 according to the sequence of ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717, which comprises 16,338 nucleotides):
GCATATAAGCAAGYACATRAYYYCTAYAVYAGTACATAAYRCATAYAATTATTRAYYGTACATAGTACATTATRTCAAAYYCATYCTYRAYARYATATYTAYYATATAYYYCYTNCCAYTAGATCACGAGCTTAAY TACCATGCCGCGTGAAACCARCAACCCGCTRRGCAGNGGATCCCTCTTCTCGCTCCGGGCCCATARAYYGTGGGGGTCGCTATYYARTGAAYTTTAYCAGGCATCTGGTTCTTTCTTCAGGGCCATCTCATCTAAARYGT CCATTCYTTCCTCTTAAATAAGACATCTCGATGGACTAATGRCTAATCAGCCCATGCTCACACATAACTGTGYTGTCATACATTTGGTATTTTTTATTTTGGGGGATGCTTGGACTCAGCTATGGCCGTCAAAGGCCCTG ACCCGGAGCATCTATTGTAGCTGGACTTAACTGCATCTTGAGCACCAGCATAATGATARGCRTG
R: A or G; Y: C or T; N: A, G, C or T
In this respect it can be seen that the Applicant has solved a certain number of technical problems for carrying out this process. Firstly, the choice of primers was a real problem, since it was necessary to select sequences which on the one hand are common to various bovine breeds and on the other hand hybridize in a stable manner under the very varied physico-chemical conditions representative of the wide diversity of organic matter which may contain biological matter of bovine origin.
The Applicant has also developed other oligonucleotide primers which are shown below and which have the characteristic of being able to generate smaller amplification fragments of a size less than about 200 base pairs and greater than 100 base pairs, and advantageously about 150 base pairs. The sequences of the oligonucleotide primers are the following:
SEQ ID No9: GAGCCTTATCAGTATTAAATTTATC (15824-15848)
SEQ ID No10: CATTAATGTTATGTACATTA (15962-15981)
SEQ ID No11: TTTCACGCGGCATGGTAATT (16162-16181)
SEQ ID No12: ATCCAATGAATTTTACCAGG (16245-16264)
SEQ ID No13: GTCAATGGTCACAGGACATA (181-200)
SEQ ID No14: ATTGACTTTGTTTGGAGTGC (319-338)
The positions of these nucleotide primers defined according to the sequence of ANDERSON et al., 1982, J. Mol. Biol., 156, 683-717 are indicated in parenthesis.
The invention also relates to pairs of oligonucleotide primers, characterized in that the oligonucleotides of which they consist are chosen from those:
having a sequence identical to the extent of at least 80%, preferably 90%, and advantageously 95%, to an oligonucleotide made up of a sequence of about 15 to 25 nucleotides, in particular 20 to 25 nucleotides, comprising at least 10 contiguous nucleotides of the following SEQ ID No. 9:
GAGCCTTATCAGTATTAAATTTATC
or of the following sequence ID No. 10:
CATTAATGTTATGTACATTA
or of the following sequence SEQ ID No. 11:
TTTCACGCGGCATGGTAATT
or of the following sequence SEQ ID No. 12:
ATCCAATGAATTTTACCAGG
or of the following sequence SEQ ID No. 13:
GTCAATGGTCACAGGACATA
or of the following sequence SEQ ID No. 14:
ATTGACTTTGTTTGGAGTGC
Each of the oligonucleotide primers SEQ ID No. 4 to 6 and SEQ ID No. 9, 12 and 13 is used as a pair with one or other of the oligonucleotide primers SEQ ID No. 1 to 3 and SEQ ID No. 10, 11 and 14. The most advantageous pairs of oligonucleotide primers are the following: SEQ ID No. 9 with SEQ ID No. 10, SEQ ID No. 6 with SEQ ID No. 11, SEQ ID No. 12 with SEQ ID No. 3, SEQ ID No. 13 with SEQ ID No. 14.
The pair of primers SEQ ID No. 9 with SEQ ID No. 10 enables the fragment advantageously having a sequence identical to the extent of at least 80%, preferably 90%, and advantageously 95%, to the following DNA fragment to be obtained:
SEQ ID No15 (positions 15824-15981): GAGCCTTATCAGTATTAAATTTATCAAAAATCCCAATAACTCAACACAGAATTTGCACCCTAACCAAATATTACAAACAC CACTAGCTAACATAACACGCCCATACACAGACCACAGAATGAATTACCTACGCAAGGGGTAATGTACATAACATTAATG
The pair of primers SEQ ID No. 6 with SEQ ID No. 11 enables the fragment advantageously having a sequence identical to the extent of at least 80%, preferably 90%, and advantageously 95%, to the following DNA fragment to be obtained:
SEQ ID No16 (positions 16029-16181): GCATATAAGCAAGTACATGACCTCTATAGCAGTACATAATACATATAATTATTGACTGTACATAGTACATTATGTCAAATTCATTCTT GATAGTATATCTATTATATATTCCTTACCATTAGATCACGAGMTAATRACCATGCCGCGTGAAA
The pair of primers SEQ ID No. 12 with SEQ ID No. 3 enables the fragment advantageously having a sequence identical to the extent of at least 80%, preferably 90%, and advantageously 95%, to the following DNA fragment to be obtained:
SEQ ID No17 (positions 16245-171): ATCCAATGAATTTTACCAGGCATCTGGTTCTTTCAGGGCCATCTCATCTAAAACGGTCCATTCTTTCCTCTTAAATAAGACATCTCGATGGACT AATGGCTAATCAGCCCATGCTCACACATAACTGTGCTGTCATACAT TTGGTATTTTTTTATTTTGGGGGATGCTTGGACTCAGCTATGGCCGTCAAAGGCCCTGACCCGGAGCATCTATT GTAGCTGGACTTAACTGCATCTTGAGCACCAGCATAATGATAAGCATG
The pair of primers SEQ ID No. 13 with SEQ ID No. 14 enables the fragment advantageously having a sequence identical to the extent of at least 80%, preferably 90%, and advantageously 95%, to the following DNA fragment to be obtained:
SEQ ID No18 positions 181-338): GTCAATGGTCACAGGACATAAATTATATTATATATCCCCCCTTCATAAAAATTCCCCCCTTAAATATCTACCACCACTTTTAACAGACTTTTCCCTAG ATACTTATTTAAATTTTTCACGCAACAATACTCAATTTAGCACTCCAAACAAAGTCAAT
The amplification products described above, and in particular the sequences SEQ ID No. 15 to SEQ ID No. 18, can be detected if a significant fraction of the DNA is degraded, that is to say after the action of the physical, chemical and/or biochemical factors described above and during transformations of organic substrates.
The experimenter preferably searches for the presence of SEQ ID No. 8, described above, with the aid of suitable primers, and in the case where the detection is negative, searches for the fragments of lower size, and in particular those of about 150 to 260 base pairs generated, in particular, by the use of the primers SEQ ID No. 9 to SEQ ID No. 14.
The use of primers SEQ ID No. 9 to SEQ ID No. 14 gives rise only to single DNA fragments of about 150 to 260 base pairs. The process described above is specific since it gives no amplification reaction which can be detected in the presence of DNA of other than bovine origin. The present, invention also relates to these oligonucleotide fragments.
The uniqueness of the amplification product is another advantage of the present invention, enabling a high sensitivity to be obtained and greatly facilitating interpretation of the results.
The process according to the present invention thus has a large number of advantages with respect to the techniques of identification of bovine DNA which are already known.
The process described thus has a high simplicity in its interpretation because of the production of a single and unique product which is specific to bovine DNA and which thus is not found in products of amplification of DNA of other species.
Reading of the migration profiles of the amplification products obtained with the process according to the present invention thus comprises simply determination of the presence of a single and unique migration band in an electrophoresis gel. In the absence of such a band, it can be considered that there are no detectable traces of bovine DNA. In contrast, the presence of a band indicates that bovine DNA is present in the sample, and thus that the sample in question contains biological matter of bovine origin.
The amplification product can be demonstrated by any method known to those skilled in the art, and in particular by simple electrophoresis on an agarose gel. This amplification product can be sequenced in order to determine the nucleotide sequence and to confirm its identity. It can also be demonstrated by hybridization with a probe comprising an oligonucleotide part and a marker. The oligonucleotide which make up this probe comprises a minimum of about 15 nucleotides, preferably a minimum of about 20 nucleotides.
To confirm the identity of the fragment SEQ ID No. 8, an oligonucleotide in which part of the sequence is identical to the extent of at least 80%, preferably 90%, to the following sequences SEQ ID No. 7 or No. 19 is used:
SEQ ID No7 (16114-16140): CTTGATAGTATATCTATTATATATTCC (BH1)
SEQ ID No19 (16227-16251): TAARCCGTGGGGGTCGCTATCCAAT (BH2)
where R is G or A;
The identity of fragments SEQ ID No. 15 to SEQ ID No. 18 will advantageously be confirmed by sequencing.
The marker can be any marker known to those skilled in the art, but is preferably digoxigenin (DIG).
The use of the probes described above to demonstrate the product of the amplification reaction SEQ ID No. 8 is particularly advantageous since it enables the bovine origin of this amplification product to be confirmed and therefore also enables the specificity and sensitivity of the process to be improved.
The present invention also relates to complementary and inverse/complementary oligonucleotides to the oligonucleotides described above.
Those skilled in the art could advantageously refer to the general manual of SAMBROOK et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., or to one of its recent re-issues, for carrying out the molecular biology techniques of the present invention.
The present invention enables the presence of compounds of bovine origin to be detected in products used in agricultural food production and in the cosmetics industry, such as cooked or raw meats, pellets and meals used for feeding cattle, composts, manures and dungs, products based on blood, bone meal, hide or guano, gelatines and animal fats.